Olfactory transduction begins with the binding of an odorant ligand to a protein receptor on the olfactory neuron cell surface, thus initiating a cascade of enzymatic reactions that results in the production of a second messenger and the eventual depolarization of the cell membrane (1,2). This relatively straightforward and common signalling motif is complicated by the fact that there are several thousand odorants, mostly low molecular weight organic molecules, and nearly one thousand different receptors (3,4). The receptors are members of the superfamily of membrane receptors characterized structurally by possessing seven transmembrane spanning helices, and functionally by being coupled to GTP-binding proteins. Other members of this superfamily recognize diverse ligands from peptides to biogenic amine neurotransmitters, hormones, drugs, and other organic compounds. The odorant receptor sub-family is the largest sub-family of G-protein coupled receptors (GPCRs) but remains in some ways the most enigmatic since no particular receptor has been definitively paired with any ligand. Strictly speaking vertebrate odorant receptors are classified as "orphan" receptors--receptors with no identified ligand (5,6).
This situation is especially problematic for understanding coding in the olfactory system and appreciating the nature of the neural image passed to the brain by the peripheral transducing cells (7). Current models of olfactory processing have been driven largely by genetic data based on the now well described patterns of receptor gene expression (8,9). Receptors can be grouped into sub-families based on sequence similarities, and subfamilies of receptors are known to be expressed within one of four restricted topographic zones in the nasal epithelium, although within these general zones expression patterns appear to be random (10,11). Further, all neurons expressing a particular receptor gene converge to a restricted target in the olfactory bulb (12, 13). However, olfactory neurons typically generate physiological responses to multiple odorants (14, 15) and if, as most evidence indicates, each cell expresses only one type of receptor (11, 16), then the receptors must be able to bind a variety of molecules. Thus, while it may be attractive to hypothesize that the genetic categorization of receptor sequences reflects systematic differences in ligand specificities, and that genetic expression patterns underlie a spatial map for odorant sensitivity, experimental validation of these ideas requires knowing the correlation between receptor gene sequence and the encoded receptor protein's binding specificities, i.e. its receptive field.
Further progress in this area has been limited by the absence of a reliable and efficient system for expressing and assaying cloned odorant receptors. There appear to be two main obstacles to obtaining odorant receptor expression in a heterologous system. For one, expressed receptors must be properly targeted to, and inserted in, the plasma membrane, a process that may require specialized cellular machinery not available in heterologous cell culture expression systems. Secondly, even properly inserted receptors must couple to a second messenger system in order to produce a response that can be assayed (17). Olfactory specific isoforms of second messenger enzymes have been identified in olfactory neurons (2), raising the possibility that receptor-effector coupling may be highly specific, and that endogenous G-proteins in heterologous cell systems may be unable to produce a powerful enough response to be measured reliably.
In order to circumvent these two potential difficulties we have adopted an alternative strategy for odorant receptor expression. On the assumption that olfactory neurons themselves would be the most capable cells for expressing, targeting and coupling odorant receptors, we used the nasal epithelium as an expression system, driving expression of a particular receptor by including it in a recombinant adenovirus (Adv) and infecting rat nasal epithelia in vivo. Adenovirus vectors have been developed as a tool for efficient gene transfer in mammalian cells (18) and have shown promise in a variety of experimental and clinical applications (19-23). Here we show that this system effectively expresses a foreign odorant receptor gene that can be conveniently assayed for specific ligand activation by physiological methods. Additionally we have been able to identify a set of ligands that activate a particular receptor. This invention provides conclusive evidence that the putative odorant receptor genes cloned several years ago do indeed encode odorant receptors and, for the first time in a vertebrate, pairs a particular receptor of known amino acid sequence with a specific set of odorant ligands.